Mannanase variants

ABSTRACT

Varients of mannanase, compositions including variants, to methods for their production to methods of using the variants to degrade and modify mannan containing material.

FIELD OF THE INVENTION

This invention relates to variants of mannanase enzyme. The variants areuseful in industrial applications wherein degradation or modification ofmannan is desired, such as in laundry and cleaning applications, infeed, food, pulp and paper and oil industry. The invention also providesuseful mannanases enzymes, polynucleotides encoding these enzymes,enzyme compositions and methods for their production and use.

BACKGROUND

Mannans are mannose containing polysaccharides found in various plants.Mannans are poorly soluble in aqueous environment and theirphysicochemical properties give rise to viscous dispersions.Additionally, mannans have high water binding capacity. All of thesecharacteristics cause problems in several industries including brewing,baking, animal nutrition, and laundry and cleaning applications.

In plant-based diets different β-mannans are present and depending ontheir amounts and properties they can compromise nutrient digestion,microbial colonisation and growth performance. Enzymatic degradation ofmannans reduces digesta viscosity of high water soluble mannans andleads to production of manno-oligosaccharides that may formwater-insoluble linear mannans present in leguminoseae. Mannanaseincreases average daily gain, feed efficiency, weight uniformity andlivability in all monogastric animals.

For animal feed applications, such as feed for monogastric animals withcereal diets, mannan is a contributing factor to viscosity of gutcontents and it thereby adversely affects the feed digestibility andanimal growth rate. For ruminants, mannan represents a substantialcomponent of fiber intake and a more complete digestion of mannan wouldfacilitate higher feed conversion efficiencies.

For laundry and cleaning applications enzyme compositions comprisingmannanase can be used to degrade mannan. However, providing mannanasesthat are stable in varying storage and use conditions while stillshowing good mannan degrading activity is difficult.

N-linked glycosylation of proteins is a type of post-translationalmodification where a sugar molecule oligosaccharide known as glycan isattached to an amide nitrogen group of an asparagine (Asn, N) residue ofa protein. This type of linkage is important for both the structure andfunction of enzymes and other proteins.

It is an object of the present invention to provide variants ofmannanase having improved stability and exhibiting mannanase activitywhen applied in different industrial processes, as well as enzymecompositions for mannan degradation or modification.

SUMMARY

According to the first aspect is provided a variant of mannanase havingmannanase activity and a non-glycosylated amino acid at the position283, wherein the variant is selected from the group consisting of

-   -   1) a polypeptide having at least 85% sequence identity to        residues 27-331 of SEQ ID NO: 2;    -   2) a polypeptide encoded by a polynucleotide that hybridizes        under high stringency conditions with        -   a) nucleotides 79-993 of SEQ ID NO: 1 (man7)        -   b) the full-length complement of a); and    -   3) a variant encoded by a polynucleotide having at least 95%        sequence identity to the SEQ ID NO: 1 or the genomic DNA        sequence thereof;        and wherein the amino acid numbering corresponds to the amino        acid numbering of SEQ ID NO: 2 (Man7) full-length amino acid        sequence containing a signal sequence.

The present variant of mannanase is advantageous in having goodstability and mannanase activity. The variant has differentglycosylation pattern compared to a wild type mannanase, which mayprovide improved specific activity, stability and yield when produced ina host cell. Thus, the present variant of mannanase may provide improvedyield in production and better performance in use. The stability of thevariant is particularly good in detergents and at temperatures typicallyused in applications where mannan degradation is used, such as inlaundry detergents.

According to the second aspect of the invention is provided an enzymecomposition comprising the variant of mannanase of the first aspect and

-   -   a. at least one preservative selected for example from group        consisting of organic acid, citric acid, ascorbic acid, benzoic        acid and their salts and derivatives, sodium benzoate, benzoate,        hydroxybenzoate and derivatives, so sorbic acid, sodium sorbate,        sorbate, salts, such as sodium chloride or potassium chloride,        1,2-Benzisothiazolin-3-one (BIT), or a combination thereof;    -   b. optionally at least one stabilizer selected from polyol,        propylene glycol, polyethylene glycol, hexylene glycol,        glycerol, a sugar, sugar alcohol, polysaccharide, lactic acid,        boric acid, boric acid derivative, aromatic borate ester,        4-formylphenyl boronic acid, phenyl boronic acid derivative,        peptide, surfactant, or a combination thereof;    -   c. optionally at least one enzyme selected from proteases,        amylases, cellulases, lipases, xylanases, mannanases, cutinases,        esterases, phytases, DNAses, pectinases, pectinolytic enzymes,        pectate lyases, carbohydrases, arabinases, galactanases,        xanthanases, xyloglucanases, laccases, peroxidases and oxidases        with or without a mediator, or a combination thereof; and    -   d. optionally at least one filler selected from maltodextrin,        flour, sodium chloride, sulfate, sodium sulfate, or a        combination thereof.

As evidenced by the Examples, the variants comprised in the enzymecomposition according to the invention have a structure and propertiesthat allow production in recombinant host cells and make them useful inenzyme compositions for industrial applications. The enzyme compositionis particularly good for detergent formulations because the variant ofmannanase has good stability, wash performance and specific activitywhen used to degrade mannan in laundry and washing use.

According to the third aspect there is provided a detergent compositioncomprising the variant of mannanase of the first aspect or the enzymecomposition of the second aspect.

The present detergent composition is advantageous in that it isefficient and economical in removing mannan containing stains.

According to another aspect is provided a use of, and a method of using,the present enzyme composition or the present variant of mannanase in adetergent.

According to the fourth aspect is provided a recombinant host cellcomprising genetic elements that allow producing at least onerecombinant polypeptide comprising the variant of mannanase of the firstaspect.

According to the fifth aspect is provided a method for producing arecombinant polypeptide having mannanase activity and comprising:

-   -   a. cultivating a recombinant host cell of the fourth aspect,        wherein        -   the genetic elements comprise at least one control sequence            which controls the production of the recombinant polypeptide            in the recombinant host cell;        -   the genetic elements optionally comprise at least one            sequence encoding a signal sequence for transporting the            recombinant polypeptide outside the host cell; and        -   cultivating is carried out in conditions allowing production            of the recombinant polypeptide; and    -   b. recovering the recombinant polypeptide.

The method provides an efficient way to produce a recombinantpolypeptide comprising a variant of mannanase. Because the variant ofmannanase is produced in a recombinant host cell, a production system isprovided which can be optimized, tailored, and controlled in a desiredmanner. The variant of mannanase produced by the method may differ fromnatural mannanases at a structural and functional level. The variant ofmannanase produced by the method has a glycosylation pattern, or otherpost translational modification, which causes differences in thestructure and/or function when compared to a natural mannanase, such asa mannanase having similar amino acid sequence, or compared to amannanase having the same amino acid sequence but produced in anotherhost cell. In particular, when the variant is produced in a host cellcapable of N-linked glycosylation, the variant has advantageously anon-glycosylated residue Asn283 resulting to improved properties. Thevariant of mannanase produced by the present method can be used as such,or formulated into a selected formulation.

According to another aspect is provided an enzyme preparation comprisinga recombinant polypeptide having mannanase activity and obtainable byusing the present host cell.

The enzyme preparation or the enzyme composition may further compriseother enzyme(s) selected from the group consisting of proteases,amylases, cellulases, lipases, xylanases, mannanases, cutinases,esterases, phytases, DNAses, pectinases, pectate lyases, pectinolyticenzymes, carbohydrases, arabinases, galactanases, xanthanases,xyloglucanases, laccases, peroxidases and oxidases with or without amediator, as well as suitable additives selected from the groupconsisting of stabilizers, buffers, surfactants, bleaching agents,mediators, anti-corrosion agents, builders, anti-redeposition agents,optical brighteners, dyes, pigments, perfumes, caustics, abrasives andpreservatives.

According to a sixth aspect is provided a method for degrading ormodifying mannan containing material comprising treating said mannancontaining material with an effective amount of the present enzymecomposition or the present variant of mannanase.

According to the seventh aspect is provided an animal feed comprisingthe present enzyme composition or the present variant of mannanase, andat least one protein source of plant origin or a mannan containingproduct or by-product, and

-   -   a. Optionally at least one enzyme selected from protease,        amylase, phytase, xylanase, endoglucanase, beta-glucanase, or a        combination thereof; and    -   b. Optionally at least one filler selected from maltodextrin,        flour, salt, sodium chloride, sulfate, sodium sulfate, or a        combination thereof.

According to the eighth aspect is provided a feed supplement comprisingthe present enzyme composition or the present variant of mannanase; and

-   -   a. Optionally at least one enzyme selected from protease,        amylase, phytase, xylanase, endoglucanase, beta-glucanase, or a        combination thereof; and    -   b. Optionally at least one filler selected from maltodextrin,        flour, salt, sodium chloride, sulfate, sodium sulfate, or a        combination thereof.

The feed and the feed supplement improve nutritional value of feedcompared to a feed without the variant. The present enzyme compositioncomprises the variant of mannanase, which has improved stability. Thepresent enzyme composition and the present variant degrade mannanpresent in the feed and thereby make it more easily digestible for theanimal. In particular for soybean meal containing feedsmannan-oligosaccharides that result from enzymatic digestion have abeneficial effects on the intestinal microbes, and consequently on theperformance of the animals. The effect of the variants of mannanase canbe enhanced by including xylanase to digest arabinoxylans present incorn soybean based diets. The present variants can also be used tomodify rheological properties of wet feeds.

In an embodiment the feed may comprise animal protein, such as meat mealor bone meal.

According to another aspect is provided a use, and a method of using,the present animal feed or the present feed supplement in:

-   -   a. feeding animals;    -   b. improving weight gain of animals.

In an embodiment the animal is a monogastric animal or a ruminant. Inanother embodiment the animal is a broiler chicken, egg-laying chicken,swine, turkey, or an aquaculture organism such as fish. In anotherembodiment the animal is a ruminant.

According to a ninth aspect is provided a use of, and a method of using,the present variant or the present enzyme composition in oil drilling orhydro-fracturing.

The present enzyme composition and the present variant are advantageousin modifying rheological properties of oil drilling fluids andhydro-fracturing fluids, and to improve oil recovery.

According to an tenth aspect is provided a use of, and a method ofusing, the present variant or the present enzyme composition inprocessing coffee extract, fruit juice, pineapple juice, or soya milk.

Using the present variant and the present enzyme composition isadvantageous in processing coffee extract because it reduces viscosityof the coffee extract.

Using the present variant and the present enzyme composition isadvantageous in processing and manufacturing fruit juice because itlowers viscosity and improves filtration rate, stability and helps toextract fruit components.

Using present variant and the present enzyme composition is advantageousin processing and manufacturing soya milk because it improves yield,colour, protein content and taste of soya milk.

In another aspect is provided a nucleic acid molecule encoding thepresent variant of mannanase.

In another aspect is provided a vector comprising the present nucleicacid molecule.

In another aspect is provided a functional fragment of the presentvariant of mannanase, and a polynucleotide encoding it.

In another aspect the disclosed sequence information herein relating toa polynucleotide sequence encoding a mannanase of the invention can beused as a tool to identify other homologous mannanases. For instance,polymerase chain reaction (PCR) can be used to amplify sequencesencoding other homologous mannanases from a variety of biologicalsources. In addition, genome mining approaches can be used to identifysequences encoding other homologous mannanases from genome databases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows schematic representation of expression cassette.

FIG. 2 shows the relative specific activity of the variants (TBH5, TBH6,TBH9, TBH10 and TBH11) compared to the wild type Man7 mannanase(produced in Trichoderma).

FIG. 3A-B describe the stain removal performance of variants and wildtype Man7 (produced in Trichoderma) as an increase of lightness (sum ofΔL*of 3 stains) in the presence of 4,4 g/l of Commercial heavy dutyliquid detergent A at 40° C., 16°dH, 60 min, pH approx. 8.3 and enzymesdosed as activity units (MNU) per wash liquor.

FIG. 3A shows variants TBH1, TBH2, TBH3, TBH4, TBH5, TBH6, TBH7, TBH8,TBH9 and wild type.

FIG. 3B shows variants TBH6, TBH10, TBH11 and wild type.

FIG. 4A-B describe the stain removal performance of variants and wildtype Man7 (produced in Trichoderma) as an increase of lightness (sum ofΔL*of 3 stains) in the presence of 3,8 g/l of Commercial color detergentpowder at 40° C., 16°dH, 60 min, pH approx. 10 and enzymes dosed asactivity units (MNU) per wash liquor.

FIG. 4A shows variants TBH1, TBH2, TBH3, TBH4, TBH5, TBH6, TBH7, TBH8,TBH9 and wild type Man7

FIG. 4B shows variants TBH6, TBH10, TBH11 and wild type Man7.

FIG. 5 describes the stain removal performance of variants TBH1, TBH2,TBH3, TBH4, TBH5, TBH6, TBH7, TBH8, TBH9 and wild type Man7 (produced inTrichoderma) as an increase of lightness (sum of ΔL*of 3 stains) in thepresence of 4,2 g/l of Commercial bleach detergent powder at 40° C.,16°dH, 60 min, pH approx. 9.5 and enzymes dosed as activity units (MNU)per wash liquor.

FIG. 6 shows a flow chart of instant coffee production involving use ofthe mannanase variants of the invention.

DEPOSITS

The following strain depositions according to the Budapest Treaty on theInternational Recognition of Deposit of Microorganisms for the Purposesof Patent Procedure were made:

The E. coli strain RF12379 including the plasmid pALK4434 was depositedat the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany on 2 Mar.,2017 and assigned accession number DSM 32425.

The E. coli strain RF12380 including the plasmid pALK4435 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany on 2 Mar.,2017 and assigned accession number DSM 32426.

The E. coli strain RF12381 including the plasmid pALK4436 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany on 2 Mar.,2017 and assigned accession number DSM 32427.

The E. coli strain RF12382 including the plasmid pALK4437 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany on 2 Mar.,2017 and assigned accession number DSM 32428.

The E. coli strain RF12383 including the plasmid pALK4438 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany on 2 Mar.,2017 and assigned accession number DSM 32429.

The E. coli strain RF12384 including the plasmid pALK4439 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany on 2 Mar.,2017 and assigned accession number DSM 32430.

The E. coli strain RF12385 including the plasmid pALK4440 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany on 2 Mar.,2017 and assigned accession number DSM 32431.

The E. coli strain RF12386 including the plasmid pALK4441 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany on 2 Mar.,2017 and assigned accession number DSM 32432.

The E. coli strain RF12387 including the plasmid pALK4442 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany on 2 Mar.,2017 and assigned accession number DSM 32433.

The E. coli strain RF12456 including the plasmid pALK4432 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany on 18 May,2017 and assigned accession number DSM 32518.

The E. coli strain RF12457 including the plasmid pALK4433 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany on 18 May,2017 and assigned accession number DSM 32519.

SEQUENCE LISTINGS

SEQ ID NO: 1 DNA sequence of the man7

SEQ ID NO: 2 Full-length amino acid sequence of the Man7

SEQ ID NO: 3 Deduced amino acid sequence (mature) of the Man7

SEQ ID NO: 4 Core amino acid sequence of the Man7, no CBM

SEQ ID NO: 5 Synthetic gene sequence of the variant tbh1

SEQ ID NO: 6 Deduced amino acid sequence (mature) of the variant TBH1

SEQ ID NO: 7 Synthetic gene sequence of the variant tbh2

SEQ ID NO: 8 Deduced amino acid sequence (mature) of the variant TBH2

SEQ ID NO: 9 Synthetic gene sequence of the variant tbh3

SEQ ID NO: 10 Deduced amino acid sequence (mature) of the variant TBH3

SEQ ID NO: 11 Synthetic gene sequence of the variant tbh4

SEQ ID NO: 12 Deduced amino acid sequence (mature) of the variant TBH4

SEQ ID NO: 13 Synthetic gene sequence of the variant tbh5

SEQ ID NO: 14 Deduced amino acid sequence (mature) of the variant TBH5

SEQ ID NO: 15 Synthetic gene sequence of the variant tbh6

SEQ ID NO: 16 Deduced amino acid sequence (mature) of the variant TBH6

SEQ ID NO: 17 Synthetic gene sequence of the variant tbh7

SEQ ID NO: 18 Deduced amino acid sequence (mature) of the variant TBH7

SEQ ID NO: 19 Synthetic gene sequence of the variant tbh8

SEQ ID NO: 20 Deduced amino acid sequence (mature) of the variant TBH8

SEQ ID NO: 21 Synthetic gene sequence of the variant tbh9

SEQ ID NO: 22 Deduced amino acid sequence (mature) of the variant TBH9

SEQ ID NO: 23 Synthetic gene sequence of the variant tbh10

SEQ ID NO: 24 Deduced amino acid sequence (mature) of the variant TBH10

SEQ ID NO: 25 Synthetic gene sequence of the variant tbh11

SEQ ID NO: 26 Deduced amino acid sequence (mature) of the variant TBH11

DETAILED DESCRIPTION

Mannan refers to polysaccharides consisting of a mannose backbone linkedtogether by β-1,4-linkages with side-chains of galactose attached to thebackbone by α-1,6-linkages. Mannans comprise plant-based material suchas guar gum and locust bean gum. Glucomannans are polysaccharides havinga backbone of more or less regularly alternating β-1,4 linked mannoseand glucose, galactomannans and galactoglucomannans are mannans andglucomannans with alpha-1,6 linked galactose side branches.

The term “functional fragment” or “effective fragment” means a fragmentor portion of SEQ ID NO: 2 (Man7) variant that retains about the sameenzymatic function or effect.

The term “mannanase variants” and “variant of mannanase” means anymannanase molecule obtained by site-directed or random mutagenesis,insertion, substitution, deletion, recombination and/or any otherprotein engineering method, which leads to mannanases that differ intheir amino acid sequence from the parent mannanase, i.e. a wild-typemannanase. The terms “wild type mannanase”, “wild type enzyme”, “wildtype”, or “wt” in accordance with the disclosure describe a mannanaseenzyme with an amino acid sequence found in nature or a fragmentthereof.

The term “catalytic activity” or “activity” describes quantitatively theconversion of a given substrate under defined reaction conditions. Theterm “residual activity” is defined as the ratio of the catalyticactivity of the enzyme under a certain set of conditions to thecatalytic activity under a different set of conditions. Therefore theresidual activity a_(i) is given by a_(i)=v_(i)/v₀ where v denotes anymeasure of catalytic activity and a_(i)*100 is the relative activity inpercent. The term “specific activity” describes quantitatively thecatalytic activity per amount of enzyme under defined reactionconditions.

The term “proteolytic stability” describes the property of a protein towithstand a limited exposure to proteases under conditions where theproteases are active, without losing activity under conditions where itsactivity can be measured.

As used herein, the term “mannanase” or “galactomannanase” denotes amannanase enzyme defined according to that known in the art as mannanendo-1,4-beta-mannosidase and having the alternative namesbeta-mannanase and endo-1,4-mannanase and catalysing hydrolysis of1,4-beta-D-mannosidic linkages in mannans, galactomannans, glucomannans,and galactoglucomannans. Mannanases are classified according to theEnzyme Nomenclature as EC 3.2.1.78.

As used herein, “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including any enzyme, variant, nucleic acid, protein, peptideor cofactor, that is at least partially removed from one or more or allof the naturally occurring constituents with which it is associated innature; (3) any substance modified by the hand of man relative to thatsubstance found in nature, such as a variant; or (4) any substancemodified by increasing or decreasing the amount of the substancerelative to other components with which it is naturally associated(e.g., recombinant production in a host cell; one or multiple copies ofa gene encoding the substance; and use of an alternative promoter to thepromoter naturally associated with the gene encoding the substance). Inan embodiment a polypeptide, enzyme, variant, polynucleotide, host cellor composition of the invention is isolated.

As used herein, the term “comprising” includes the broader meanings of“including”, “containing”, and “comprehending”, as well as the narrowerexpressions “consisting of” and “consisting only of”.

As used herein, “variant” means a sequence or a fragment of a sequence(nucleotide or amino acid) inserted, substituted or deleted by one ormore nucleotides/amino acids, or which is chemically modified. In anembodiment the term variant also includes a recombinant mannanaseenzyme.

As used herein, “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. In an embodiment the conservativeamino acids in the present description refer to the amino acids withinfollowing groupings: Hydrophobic (F W Y H KM I L V A G C); Aromatic (F WY H); Aliphatic (I L V); Polar (W Y H K R E D C S T N Q); Charged (H K RE D); Positively charged (H K R); Negatively charged (E D); Small (V C AG S P T N D); Tiny (A G S). Thus, a conservative substitution occurswhen an amino acid is substituted with an amino acid in the same group.

In an embodiment the substitution is a substitution with at least oneamino acid residue. In a further embodiment the at least amino acid isAla.

As used herein, a “non-conservative amino acid substitution” is one inwhich an amino acid is substituted with an amino acid in a differentgroup as defined above. The non-conservative substitution may resultinto a change of an amino acid to another amino acid with differentbiochemical properties, such as charge, hydrophobicity and/or size. Inan embodiment the non-conservative substitution changes at least oneproperty of the variant, such as stability, glycosylation pattern,folding, structure, activity, or affinity.

N-linked glycosylation process occurs in eukaryotes, but very rarely inbacteria. The attachment of a glycan residue to a protein requires therecognition of a consensus sequence. N-linked glycans are almost alwaysattached to an asparagine (Asn) side chain that is present as a part ofAsn-X-Ser/Thr consensus sequence, wherein X is any amino acid except forproline (Pro). The inventors have found that a non-glycosylated Asn sidechain structurally close to the active site of the mannanase isimportant for obtaining a variant with good mannan degradingperformance. Without being bound to any theory, glycan sugars are polarmolecules and when attached to an Asn, they locate on the surface of theprotein causing structural changes in the glycosylated Asn and in itsvicinity. Site directed mutagenesis of Asn or Ser/Thr residues in theAsn-Xaa-Thr(Ser) consensus sequence can be used to prevent glycosylationof the desired N-linked glycosylation sites in the variant of the firstaspect of the invention.

In an embodiment the variant has a substitution of an amino acid by aresidue which prevents N-linked glycosylation of the residue 283 whenexpressed in a host cell capable of N-linked glycosylation.

In an embodiment the present variant comprises at least oneAsn-X-Ser/Thr consensus sequence.

In an embodiment the present variant comprises Pro residue in thelocation X of the at least one Asn-X-Ser/Thr consensus sequence.

In an embodiment the substitution is either a conservative or anon-conservative substitution.

As used herein, a “peptide” and a “polypeptide” are amino acid sequencesincluding a plurality of consecutive polymerized amino acid residues.For purpose of this invention, peptides are molecules including up to 20amino acid residues, and polypeptides include more than 20 amino acidresidues. The peptide or polypeptide may include modified amino acidresidues, naturally occurring amino acid residues not encoded by acodon, and non-naturally occurring amino acid residues. As used herein,a “protein” may refer to a peptide or a polypeptide of any size. Aprotein may be an enzyme, a protein, an antibody, a membrane protein, apeptide hormone, regulator, or any other protein.

The term “polynucleotide” denotes a single- or double-stranded polymerof deoxyribonucleotide or ribonucleotide bases read from the 5′ to the3′ end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules.

As used herein, “modification”, “modified”, and similar terms in thecontext of polynucleotides refer to modification in a coding or anon-coding region of the polynucleotide, such as a regulatory sequence,5′ untranslated region, 3′ untranslated region, up-regulating geneticelement, down-regulating genetic element, enhancer, suppressor,promoter, exon, or intron region. The modification may in someembodiments be only structural, having no effect on the biologicaleffect, action or function of the polynucleotide. In other embodimentsthe modification is a structural modification, which provides a changein the biological effect, action or function of the polynucleotide. Sucha modification may enhance, suppress or change the biological functionof the polynucleotide.

As used herein, “identity” means the percentage of exact matches ofamino acid residues between two aligned sequences over the number ofpositions where there are residues present in both sequences. When onesequence has a residue with no corresponding residue in the othersequence, the alignment program allows a gap in the alignment, and thatposition is not counted in the denominator of the identity calculation.Identity is a value determined with the Pairwise Sequence Alignment toolEMBOSS Needle at the EMBL-EBI website(www.ebi.ac.uk/Tools/psa/emboss.needle/).

As used herein, low stringency conditions mean for probes of at least100 nucleotides in length conditions corresponding to hybridizing atprehybridisation and hybridisation at 55° C. in 5×SSC, 0.1%N-lauroylsarcosine, 0.02% SDS, 1% blocking reagent (Roche 11 096 176001), following standard Southern blotting procedures for 12 to 24hours. The carrier material is finally washed two to three times eachfor 15 minutes using 2×SSC, 0.1% SDS at 55° C.

As used herein, high stringency conditions mean for probes of at least100 nucleotides in length conditions corresponding to hybridizing atprehybridisation and hybridization at 65° C. in 5×SSC, 0.1%N-lauroylsarcosine, 0.02% SDS, 1% blocking reagent (Roche 11 096 176001), following standard Southern blotting procedures for 12 to 24hours. The carrier material is finally washed two to three times eachfor 15 minutes using 0.1×SSC, 0.1% SDS at 65° C.

As used herein, “host cell” means any cell type that is susceptible totransformation, transfection, transduction, mating, crossing or the likewith a nucleic acid construct or expression vector comprising apolynucleotide. The term “host cell” encompasses any progeny that is notidentical due to mutations that occur during replication. Non-limitingexamples of a host cell are fungal cells, filamentous fungal cells fromDivision Ascomycota, Subdivision Pezizomycotina; preferably from thegroup consisting of members of the Class Sordariomycetes, SubclassHypocreomycetidae, Orders Hypocreales and Microascales and Aspergillus,Chrysosporium, Myceliophthora and Humicola; more preferably from thegroup consisting of Families Hypocreacea, Nectriaceae, Clavicipitaceae,Microascaceae, and Genera Trichoderma (anamorph of Hypocrea), Fusarium,Gibberella, Nectria, Stachybotrys, Claviceps, Metarhizium, Villosiclava,Ophiocordyceps, Cephalosporium, and Scedosporium, more preferably fromthe group consisting of Trichoderma reesei (Hypocrea jecorina), T.citrinoviridae, T. longibrachiatum, T. virens, T. harzianum, T.asperellum, T. atroviridae, T. parareesei, Fusarium oxysporum, F.gramineanum, F. pseudograminearum, F. venenaturn, Gibberella fujikuroi,G. moniliformis, G. zeaea, Nectria (Haematonectria) haematococca,Stachybotrys chartarum, S. chlorohalonata,

Claviceps purpurea, Metarhizium acridum, M. anisopliae, Villosiclavavirens, Ophiocordyceps sinensis, Acremonium (Cephalosporium)chrysogenum, and Scedosporium apiospermum, and Aspergillus niger,Aspergillus awamori, Aspergillus oryzae, Chrysosporium lucknowense,Myceliophthora thermophila, Humicola insolens, and Humicola grisea, mostpreferably Trichoderma reesei. Non-limiting examples of a host cell arebacterial cells, preferably gram positive Bacilli (e.g. Bacillussubtilis, B. licheniformis, B. megaterium, B. amyloliquefaciens, B.pumilus), gram-negative bacteria (e.g. Escherichia coli),actinomycetales (e.g. Streptomyces sp.) and yeasts (e.g. Saccharomycescerevisiae, Pichia pastoris, Yarrowia lipolytica).

In an embodiment the host cell is a fungal cell, preferably afilamentous fungal cell, such as Trichoderma or Trichoderma reesei. Inan embodiment the host cell is a bacterial cell, preferably a grampositive Bacillus cell, such as B. subtilis, B. licheniformis, B.megaterium, B. amyloliquefaciens, B. pumilus.

In an embodiment the host cell is capable of N-linked glycosylation.

A “recombinant cell” or “recombinant host cell” refers to a cell or hostcell, which has been genetically modified or altered to comprise anucleic acid sequence which is not native to said cell or host cell. Thegenetic modification may comprise integrating the polynucleotide in thegenome of the host cell. The polynucleotide may also be exogenous in thehost cell. In an embodiment the present host cell is a recombinant hostcell.

As used herein, “expression” includes any step involved in theproduction of a polypeptide in a host cell including, but not limitedto, transcription, translation, post-translational modification, andsecretion. Expression may be followed by harvesting, i.e. recovering,the host cells or the expressed product.

The term “expression vector” denotes a DNA molecule, linear or circular,that comprises a segment encoding a polypeptide of interest operablylinked to additional segments that provide for its transcription. Suchadditional segments may include promoter and terminator sequences, andmay optionally include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, carrier andthe like. Expression vectors are generally derived from plasmid or viralDNA, or may contain elements of both. The expression vector may be anyexpression vector that is conveniently subjected to recombinant DNAprocedures, and the choice of vector will often depend on the host cellinto which the vector is to be introduced. Thus, the vector may be anautonomously replicating vector, i.e. a vector, which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated. In an embodiment the present vector is anexpression vector.

The term “recombinant produced” or “recombinantly produced” used hereinin connection with production of a polypeptide or protein is definedaccording to the standard definition in the art.

The term “obtained from” and “obtainable” as used herein in connectionwith a specific microbial source means that the polynucleotide isexpressed by the specific source (homologous expression), or by a cellin which a gene from the source has been inserted (heterologousexpression).

The term “enzyme composition” means either a conventional enzymaticfermentation product, possibly isolated and purified, from a singlespecies of a microorganism, such preparation usually comprising a numberof different enzymatic activities; or a mixture of monocomponentenzymes, preferably enzymes derived from bacterial or fungal species byusing conventional recombinant techniques, which enzymes have beenfermented and possibly isolated and purified separately and which mayoriginate from different species, preferably fungal or bacterial speciesor the fermentation product of a microorganism which acts as a host cellfor production of a recombinant mannanase, but which microorganismsimultaneously produces other enzymes.

The term “operably linked”, when referring to DNA segments, denotes thatthe segments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in the promoter andproceeds through the coding segment to the terminator.

The term “promoter” denotes a portion of a gene containing DNA sequencesthat provide for the binding of RNA polymerase and initiation oftranscription. Promoter sequences are commonly, but not always, found inthe 5′ non-coding regions of genes.

The term “secretory signal sequence” or “signal sequence” denotes a DNAsequence that encodes a polypeptide (a “secretory peptide”) that, as acomponent of a larger polypeptide, directs the larger polypeptidethrough a secretory pathway of a host cell in which it is produced. Thesecretory signal sequence can be native or it can be replaced withsecretory signal sequence or carrier sequence from another source.Depending on the host cell, the larger peptide may be cleaved to removethe secretory peptide during transit through the secretory pathway.

The term “core region” or “catalytic domain” denotes a domain of anenzyme, which may or may not have been modified or altered, but whichhas retained at least part of its original activity. The core region ofa mannanase according to the invention corresponds to the amino acidsaligned with the amino acids 27-331 of Man7, SEQ ID NO: 2.

By the term “linker” or “spacer” is meant a polypeptide comprising atleast two amino acids which may be present between the domains of amultidomain protein, for example an enzyme comprising an enzyme core anda binding domain such as a carbohydrate binding module (CBM) or anyother enzyme hybrid, or between two proteins or polypeptides produced asa fusion polypeptide, for example a fusion protein comprising two coreenzymes. For example, the fusion protein of an enzyme core with a CBM isprovided by fusing a DNA sequence encoding the enzyme core, a DNAsequence encoding the linker and a DNA sequence encoding the CBMsequentially into one open reading frame and expressing this construct.

Efficient amount means an amount, which is sufficient to degrade mannosein the selected application.

The following abbreviations are used for amino acids:

-   -   A Ala Alanine    -   C Cys Cysteine    -   D Asp Aspartic acid    -   E Glu Glutamic acid    -   F Phe Phenylalanine    -   G Gly Glycine    -   H His Histidine    -   I Ile Isoleucine    -   K Lys Lysine    -   L Leu Leucine    -   M Met Methionine    -   N Asn Asparagine    -   P Pro Proline    -   Q Gln Glutamine    -   R Arg Arginine    -   S Ser Serine    -   T Thr Threonine    -   V Val Valine    -   W Trp Tryptophan    -   Y Tyr Tyrosine

Substitutions are described using of the following nomenclature: aminoacid residue in the protein scaffold; position; substituted amino acidresidue(s). According to this nomenclature the substitution of, forinstance, a serine residue for a glycine residue at position 20 isindicated as Ser20Gly or S20G.

The terms “detergent composition” and “detergent” include, unlessotherwise indicated, solid, granular or powder-form all-purpose orheavy-duty washing agents, especially cleaning detergents; liquid, gelor paste-form all-purpose washing agents, especially the so- calledheavy-duty liquid (HDL) types; liquid fine-fabric detergents; handdishwashing agents or light duty dishwashing agents, especially those ofthe high-foaming type; machine dishwashing agents, including the varioustablet, granular, liquid and rinse-aid types for household andinstitutional use; liquid cleaning and disinfecting agents, car orcarpet shampoos, bathroom cleaners; metal cleaners; as well as cleaningauxiliaries such as bleach additives and “stain-stick” or pre-treattypes. The terms “detergent”, “detergent composition” and “detergentformulation” are used in reference to mixtures, which are intended foruse in a wash medium for the cleaning of soiled objects. In someembodiments, the term is used in reference to laundering fabrics and/orgarments (e.g., “laundry detergents”). In alternative embodiments, theterm refers to other detergents, such as those used to clean dishes,cutlery, etc. (e.g., “dishwashing detergents”). It is not intended thatthe present invention be limited to any particular detergent formulationor composition. It is intended that in addition to the mannanasesaccording to the invention, the term encompasses detergents that maycontain e.g., surfactants, builders, chelators or chelating agents,bleach system or bleach components, polymers, fabric conditioners, foamboosters, suds suppressors, dyes, perfume, tannish inhibitors, opticalbrighteners, bactericides, fungicides, soil suspending agents,anticorrosion agents, hydrotropes, fabric hueing agents, dispersants,dye transfer inhibiting agents, fluorescent whitening agents, soilrelease polymers, anti-redepositions agents, anti-shrink agents,anti-wrinkling agents, bactericides, binders, carriers, dyes, enzymestabilizers, fabric softeners, fillers, foam regulators, perfumes,pigments, sod suppressors, solvents, and structurants for liquiddetergents, structure elasticizing agents, enzyme inhibitors orstabilizers, enzyme activators, transferase(s), hydrolytic enzymes,oxido reductases, bluing agents and fluorescent dyes, antioxidants, andsolubilizers.

The term “textile” means any textile material including yarns, yarnintermediates, fibers, non-woven materials, natural materials, syntheticmaterials, and any other textile material, fabrics made of thesematerials and products made from fabrics (e.g., garments, linen andother articles). The textile or fabric may be in the form of knits,wovens, denims, non-wovens, felts, yarns, and towelling. The textile maybe cellulose based, such as natural cellulosics including cotton,flax/linen, jute, ramie, sisal or coir or manmade cellulosics (e.g.originating from wood pulp) including viscose/rayon, ramie, celluloseacetate fibers (tricell), lyocell or blends thereof. The textile orfabric may also be non-cellulose based such as natural polyamidesincluding wool, camel, cashmere, mohair, rabbit and silk or syntheticpolymer such as nylon, aramid, polyester, acrylic, polypropylen andspandex/elastane, or blends thereof as well as blend of cellulose basedand non-cellulose based fibers. Examples of blends are blends of cottonand/or rayon/viscose with one or more companion material such as wool,synthetic fibers (e.g. polyamide fibers, acrylic fibers, polyesterfibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyurethane fibers, polyurea fibers, aramid fibers), andcellulose-containing fibers (e.g. rayon/viscose, ramie, flax/linen,jute, cellulose acetate fibers, lyocell). Fabric may be conventionalwashable laundry, for example stained household laundry. When the termfabric or garment is used it is intended to include the broader termtextiles as well.

The term “stability” includes storage stability and stability duringuse, e.g. during a wash process (in wash stability) and reflects thestability of the mannanase according to the invention as a function oftime, e.g. how much activity is retained when the mannanase is kept insolution, in particular in a detergent solution. The stability isinfluenced by many factors, e.g. pH, temperature, detergent compositione.g. proteases, stabilizers, builders, surfactants etc. The mannanasestability may be measured using the ‘activity assay’ as described inexamples.

“Mannanase activity” as used herein refers to the mannan degradingactivity of a polypeptide. Degrading or modifying as used herein meansthat mannose units are hydrolyzed from the mannan polysaccharide by themannanase. The mannan degrading activity of the polypeptides accordingto present invention can be tested according to standard test proceduresknown in the art. Example 4 provides an example of a standard method fordetermining mannanase activity.

In an embodiment the present variant has at least position N283, T285,or S285; preferably T285 or S285; most preferably T285; substituted by aresidue, which prevents N-linked glycosylation of the residue 283 whenexpressed in a host cell capable of N-linked glycosylation.

In an embodiment the present variant has at least one furthersubstitution at a position corresponding to the position below:

-   -   229, 283, 285, 300, 340, 400, 419, 433 or 446; or S229, N283,        T285, S285, N300, N340, S400, N419, S433 or N446.

In an embodiment the substitution comprises a substitution at saidposition to an amino acid selected from Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, andVal. Preferably the substitution is a substitution to an amino acidother than Ser or Thr.

In an embodiment the variant comprises a P residue in the position 284and/or 286. A substitution to a P residue in any or both of the abovepositions may cause a structural change in the variant, which preventsN-linked glycosylation.

In an embodiment in the present variant the position T285 or S285 issubstituted to a residue other than T or S.

In an embodiment in the present variant the position T285 or S285 issubstituted to an alanine.

In an embodiment the substitution is a conservative or non-conservativesubstitution resulting into an altered glycosylation of the variant whenproduced in a host cell capable of glycosylation, preferably N-linkedglycosylation.

In an embodiment is provided a variant of mannanase comprising apolypeptide having at least 85% sequence identity to residues 27-331 ofSEQ ID NO: 2, and a non-glycosylated Asn residue at position 283 whenproduced in a host cell capable of N-linked glycosylation.

In an embodiment the present variant comprises at least one additionalglycosylated N site, wherein the position corresponding to the N283 isnot glycosylated. Such a variant is obtainable by producing the variantin a host cell which is capable of N-glycosylation, resulting into atleast partial glycosylation of the other N-glycosylation sites, butwherein the N-glycosylation of N283 is inhibited.

In a further embodiment the present variant does not comprise anyglycosylated N site. Such a variant is obtainable by producing thevariant in a host cell which is not capable of N-glycosylation, or byproducing the variant in a host cell capable of glycosylation butwherein the variant has been engineered such that the otherN-glycosylation sites are not glycosylated, resulting into inhibitedglycosylation of the variant.

In an embodiment the variant has a predicted molecular weight between50000 and 51000, preferably between 50750 and 50970, without includingthe signal sequence.

In an embodiment the variant has a predicted pl between 4.5 and 4.8,preferably between 4.6 and 4.75.

Prediction of pl or molecular weight of the variant can be carried outas described in the Table 3.

In a further embodiment of the invention the variant has mannanaseactivity. In an embodiment the variant has increased specific activitycompared to a wild type mannanase when produced in a eukaryotic hostcell. The mannanases comprised in the present enzyme composition of theinvention are suitable for degrading and modifying mannan containingmaterial in various chemical environments, preferably in detergentcompositions.

In an embodiment of the first aspect the present enzyme composition isin the form of a liquid composition or a solid composition such assolution, dispersion, paste, powder, granule, granulate, coatedgranulate, tablet, cake, crystal, crystal slurry, gel or pellet.

The present invention furthermore relates to different uses of thepresent enzyme composition, such as for degrading mannan and for use ina laundry process.

The present enzyme composition can also be used in cleaning agents orboosters that are added on top of the detergent during or before thewash and that are for example in the form of liquid, gel, powder,granules or tablets. Enzyme composition and detergent components mayalso be soaked in a carrier like textiles.

In one embodiment of the present invention the enzyme compositionfurther comprises one or more additional enzymes selected from the groupconsisting of protease, lipase, cutinase, amylase, carbohydrase,cellulase, pectinase, pectatelyase, pectinolytic enzyme, esterase,phytase, mannanase, arabinase, galactanase, xylanase, oxidase,xanthanase, xyloglucanase, DNAse, laccase, and/or peroxidase, preferablyselected from the group consisting of proteases, amylases, cellulasesand lipases.

The present enzyme composition comprising mannanase and an additionalenzyme is advantageous in providing synergistic effect. Such additionalenzymes are desired when the present enzyme composition comprisingmannanase is used in detergents e.g. when washing stains. Particularlyadvantageous synergistic enzymes that work with mannanase in detergentsare amylases, proteases and cellulases, or a combination thereof, suchas a composition comprising mannanase, amylase and protease.

In an embodiment the present detergent composition is in a form of abar, a homogenous tablet, a tablet having two or more layers, a pouchhaving one or more compartments, a regular or compact powder, bar,tablet, a granule, granulate, liquid, granulate, a paste, a gel, or aregular, compact or concentrated liquid. In one embodiment the detergentcomposition can be a laundry detergent composition, preferably a liquidor solid laundry detergent composition.

In one embodiment of the present invention the present detergentcomposition further comprises one or more additional enzyme selectedfrom the group consisting of protease, lipase, cutinase, amylase,carbohydrase, cellulase, pectinase, pectatelyase, pectinolytic enzyme,esterase, mannanase, arabinase, galactanase, xylanase, oxidase,xanthanase, xyloglucanase, laccase, DNAse and/or peroxidase, preferablyselected from the group consisting of proteases, amylases, cellulasesand lipases.

The present invention furthermore relates to the use of, and a method ofusing, the enzyme composition or the detergent composition as hereindisclosed for degrading mannan.

In a further embodiment the present invention relates to the use of, anda method of using, the enzyme composition or the detergent compositionas herein disclosed in a laundry process.

The present invention furthermore relates to a method for removing astain from a surface, comprising contacting the surface with the enzymecomposition or the detergent composition as herein disclosed.

The present invention also relates to a method for degrading mannancomprising applying the enzyme composition or the detergent compositionas herein disclosed to mannan, preferably wherein the mannan is on asurface of a textile, or at least partially embedded in a textile.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

A composition for use in solid laundry detergent, for example, mayinclude 0.000001%-5%, such as 0.000005-2%, such as 0.00001%-1%, such as0.00001%-0.1% of enzyme protein by weight of the composition.

A composition for use in laundry liquid, for example, may include0.000001%-3%, such as 0.000005%-1%, such as 0.00001%-0.1% of enzymeprotein by weight of the composition.

A composition for use in automatic dishwash, for example, may include0.000001%-5%, such as 0.000005%-2%, such as 0.00001%-1%, such as0.00001%-0.1% of enzyme protein by weight of the composition.

The additional components a-d of the second aspect of the inventionprovide improved properties for the present enzyme composition. Theenzyme composition is compatible with the additional components andimproves applicability of the enzyme composition in various uses.

Salts, such as sodium chloride and sodium sulfate function as dryingaids.

In an embodiment the variant of mannanase comprises a core region.

In an embodiment the variant of mannanase comprises a CBM.

Providing mannanases that retain activity in temperatures above ambienttemperature is advantageous for applications wherein mannan degradationis required in such conditions. Further, the mannanases according toinvention may have good stability and activity in alkaline conditions,which is advantageous in detergent use and in biomass processing.

In an embodiment the variant of mannanase has an amino acid sequencewith at least, or about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2.

In an embodiment the variant of mannanase has an amino acid sequencewith at least 90% sequence identity to SEQ ID NO:2.

In an embodiment the mannanase enzyme has an amino acid sequence whichis not 100% identical to SEQ ID NO: 2.

In an embodiment of the third aspect the host cell is selected from thegroup consisting of:

fungal cells,

-   -   filamentous fungal cells from Division Ascomycota, Subdivision        Pezizomycotina; preferably from the group consisting of members        of the Class Sordariomycetes, Subclass Hypocreomycetidae, Orders        Hypocreales and Microascales and Aspergillus, Chrysosporium,        Myceliophthora and Humicola;    -   more preferably from the group consisting of Families        Hypocreacea, Nectriaceae, Clavicipitaceae, Microascaceae, and        Genera Trichoderma (anamorph of Hypocrea), Fusarium, Gibberella,        Nectria, Stachybotrys, Claviceps, Metarhizium, Villosiclava,        Ophiocordyceps, Cephalosporium, and Scedosporium;    -   more preferably from the group consisting of Trichoderma reesei        (Hypocrea jecorina), T. citrinoviridae, T. longibrachiatum, T.        virens, T. harzianum, T. asperellum, T. atroviridae, T.        parareesei, Fusarium oxysporum, F. gramineanum, F.        pseudograminearum, F. venenatum, Gibberella fujikuroi, G.        moniliformis, G. zeaea, Nectria (Haematonectria) haematococca,        Stachybotrys chartarum, S. chlorohalonata, Claviceps purpurea,        Metarhizium acridum, M. anisopliae, Villosiclava virens,        Ophiocordyceps sinensis, Acremonium (Cephalosporium)        chrysogenum, and Scedosporium apiospermum, and Aspergillus        niger, Aspergillus awamori, Aspergillus oryzae, Chrysosporium        lucknowense, Myceliophthora thermophila, Humicola insolens, and        Humicola grisea, bacterial cells, preferably gram positive        Bacilli such as B. subtilis, B. licheniformis, B. megaterium, B.        amyloliquefaciens, B. pumilus, gram negative bacteria such as        Escherichia coli, actinomycetales such as Streptomyces sp., and    -   yeasts, such as Saccharomyces cerevisiae, Pichia pastoris,        Yarrowia lipolytica,    -   most preferably Trichoderma reesei.

In an embodiment the host cell is an eukaryotic host cell capable ofN-linked glycosylation. In a preferred embodiment the host cell isTrichoderma reesei.

The recombinant host cell can be used to produce the variant ofmannanase and to carry a polynucleotide encoding it. The recombinanthost cell is useful also in preparation of variants of mannanase withdifferent properties. For example, a host cell can be selected, whichprovides post-translational modifications beneficial for stability oractivity, or which facilitates post-processing and formulation of thevariant of mannanase produced in the host cell.

In an embodiment the present enzyme composition comprises therecombinant host cell of the second aspect.

In an embodiment the present variant of mannanase is a recombinantpolypeptide, which is a fusion protein.

In an embodiment the present recombinant polypeptide is a fusionprotein, which further comprises at least one of:

-   -   an amino acid sequence providing a secretory signal sequence;    -   an amino acid sequence which facilitates purification, such as        an affinity tag, His-tag;    -   an amino acid sequence which enhances production, such as an        amino acid sequence which is a carrier, such as CBM;    -   an amino acid sequence having an enzyme activity; and    -   an amino acid sequence providing for the fusion protein with        binding affinity, such as a carbohydrate binding moiety.

The CBM, carbohydrate binding moiety, as a carrier is advantageous e.g.in Trichoderma production.

In an embodiment the host cell is non-pathogenic. This is particularlyadvantageous for using the host cell in feed, and in detergentapplications such as in home laundry detergents.

In an embodiment of the fifth aspect the mannan containing material isselected from plant based material, textile, waste water, sewage, oil ora combination thereof.

In an embodiment the mannan containing material is textile material orfabric.

In another embodiment the mannan containing material is recycled wastepaper; mechanical pulp, chemical pulp, semi chemical pulp, Kraft orother paper-making pulps; fibres subjected to a retting process; or guargum or locust bean gum containing material.

In another embodiment degradation or modifying is carried out in anaqueous environment wherein mannanase shows activity.

In a preferred embodiment the mannan containing material, which isdegraded or modified in the method, is on a textile or a fabricoptionally with mannan stains. By degrading mannan attached to thetextile or fabric, dirt or soil bound to mannan is released and notcapable of binding again to the mannan or mannan stains. The textile orfabric can be of any material, for example cotton, flax/linen, jute,ramie, sisal or coir or manmade cellulosics (e.g. originating from woodpulp) including viscose/rayon, modal, cellulose acetate fibers(tricell), lyocell, cupro or blends thereof.

In an embodiment the present feed comprises or consists of maize andsoybean meal.

In an embodiment the protein source of plant origin comprises or consistof soy, cereal such as barley, wheat, rye, oats, or maize.

In an embodiment the mannan containing product or by-product comprisesor consists of palm kernel, guar meal or copra meal.

In an embodiment the present animal feed or the present feed supplementis formulated in the form of a wet composition or a dry composition.

In an embodiment the composition comprising at least one variant ofmannanase enzyme is used in pulp and paper industry, biobleaching, fibermodification, drainage improvement and in the oil industry, i.e. in oildrilling or oil-servicing industry for hydro-fracturing or controllingthe viscosity of drilling fluids.

In an embodiment the composition comprising at least one variant ofmannanase is used in textile and detergent industry, biomass processingand biomass hydrolysis, preferably in biofuel, starch, pulp and paper,food, baking, feed or beverage industries.

In an embodiment the variant of mannanase hydrolysesendo-beta-1,4-mannosidic linkages randomly.

In an embodiment the variant of mannanase, or the nucleotide sequenceencoding the corresponding wild type mannanase, is obtainable orderivable from a bacterial source.

In an embodiment the variant of mannanase is fused with at least onefurther polypeptide, thus forming a fusion polypeptide. The fusionpolypeptide or the further polypeptide may have other catalytic orbinding activities in addition to those of mannanase. In an embodimentthe further polypeptide comprises or consists of carbohydrate bindingmodule, which is optionally a fragment of another protein or enzymederived from the same or different organism as the mannanase.

In an embodiment the variant of mannanase is connected to the furtherpolypeptide with a linker.

In an embodiment is provided a process for machine treatment of fabricswhich process comprises treating fabric during a washing cycle of amachine washing process with a washing solution containing the variantof mannanase of the first aspect or the enzyme composition of the secondaspect.

In an embodiment is provided a use of the enzyme composition of thesecond aspect or the variant of mannanase of the first aspect, togetherwith an enzyme selected from protease, amylase, cellulase, lipase,xylanase, mannanase, cutinase, esterase, phytase, DNAse, pectinase,pectinolytic enzyme, pectate lyase, carbohydrase, arabinase,galactanase, xanthanase, xyloglucanase, laccase, peroxidase and oxidasewith or without a mediator in a cleaning composition for fabric cleaningand/or fabric stain removal.

In an embodiment is provided a use of the variant of mannanase of thefirst aspect, or the enzyme composition of the second aspect, togetherwith an enzyme selected from protease, amylase, cellulase, lipase,xylanase, mannanase, cutinase, esterase, phytase, DNAse, pectinase,pectinolytic enzyme, pectate lyase, carbohydrase, arabinase,galactanase, xanthanase, xyloglucanase, laccase, peroxidase and oxidasewith or without a mediator in a cleaning composition for cleaning hardsurfaces such as floors, walls, bathroom tile and the like.

In an embodiment is provided a use of the variant of mannanase of thefirst aspect, or the enzyme composition of the second aspect, togetherwith an enzyme selected from protease, amylase, cellulase, lipase,xylanase, mannanase, cutinase, esterase, phytase, DNAse, pectinase,pectinolytic enzyme, pectate lyase, carbohydrase, arabinase,galactanase, xanthanase, xyloglucanase, laccase, peroxidase and oxidasewith or without a mediator in a cleaning composition for hand andmachine dishwashing.

EXAMPLES

The following examples are provided to illustrate various aspects of thepresent invention. They are not intended to limit the invention, whichis defined by the accompanying claims.

Example 1 Variant Design

To improve the stability and specific activity of the wild type Man7mannanase, variant sets were designed based on structural analysis ofwild type enzyme. The structural model of Man7 was created usingBioluminate software (Schrödinger LCC) with coordinates from Bacillussp. Endo-Beta-D-1,4-Mannanase (1WKY). The design included two or morespecific mutations per variant. Table 1 shows list of variants. Theamino acid numbering corresponds to the amino acid numbering of SEQ IDNO: 2 (Man7) full length amino acid sequence containing a signalsequence.

TABLE 1 List of Man7 variants Variant: Mutations TBH1: M123I, S229A,G272Q, T285A TBH2: A158S, S229A, T285A, T307R TBH3: S229A, T285A, L316KTBH4: M123I, A158S, S229A, G272Q, T285A, T307R TBH5: M123I, S229A,G272Q, T285A, L316K TBH6: A158S, S229A, T285A, T307R, L316K TBH7: F180L,S229A, T285A, L316K TBH8: S229A, T285A TBH9: S229A, T285A, N300Q, N340Q,S400A, N419A, S433A, N446Q TBH10: A158S, S229A, T307R, L316K TBH11:A158S, T285A, T307R, L316K

Example 2 Cloning of Synthetic Mannanase Variant Genes

Standard molecular biology methods were used in the isolation and enzymetreatments of DNA (e.g. isolation of plasmid DNA, digestion of DNA toproduce DNA fragments), in E. coli transformations, sequencing etc. Thebasic methods used were as described by the enzyme, reagent or kitmanufacturer.

Variants tbh1-tbh11 were ordered from GenScript as synthetic constructswithout their own signal peptide encoding sequences and with codonoptimization for Trichoderma reesei. Plasmid DNAs obtained fromGenScript including the genes tbh1-tbh11 were resuspended in sterilewater, digested with NruI and BamHI restriction enzymes (Thermo FisherScientific) according to manufacturer's instructions and cloned into anexpression vector cleaved with NruI and BamHI. Ligation mixtures weretransformed into Escherichia coli XL1-Blue or XL10-Gold cells (AHDiagnostics) and plated on LB (Luria-Bertani) plates containing 50-100μg/ml ampicillin. Several E. coli colonies were collected from theplates and DNA was isolated with GenJet Plasmid Miniprep Kit (ThermoFisher Scientific). Positive clones were screened using restrictiondigestions and they were shown to contain inserts of expected sizes. Thefusion sites to the expression plasmid of tbh1-tbh11 mannanase geneswere sequenced and the plasmids were named pALK4415-pALK4423, pALK4430and pALK4431, respectively (For details see Example 4). The plasmid DNAsincluding the tbh genes delivered by GenScript were also transformedinto XL10-Gold E. coli cells (Agilent) and deposited into DSMZ straincollection. The relevant information on the genes and the deduced aminoacid sequences (SEQ ID NOs: 5-26) are summarized in Table 2 and Table 3,respectively. The E. coli strains RF12379-RF12387, RF12456 and RF12457including the plasmids pALK4434-pALK4442, pALK4432 and pALK4433,respectively were deposited to the DSMZ collection under the accessionnumbers DSM 32425, DSM 32426, DSM 32427, DSM 32428, DSM 32429, DSM32430, DSM 32431, DSM 32432, DSM32433, DSM 32518 and DSM 32519,respectively.

TABLE 2 The summary on the mannanase variant encoding synthetic genestbh1-tbh11 Gene Length (bp)^((a) SEQ ID NO tbh1 1395 5 tbh2 1395 7 tbh31395 9 tbh4 1395 11 tbh5 1395 13 tbh6 1395 15 tbh7 1395 17 tbh8 1395 19tbh9 1395 21 tbh10 1395 23 tbh11 1395 25 ^((a)The STOP codon is included

TABLE 3 The summary of the amino acid sequences deduced from themannanase variant encoding gene sequences. Predicted Predicted (Da), pl,SEQ Man No of Length Core ss not ss not ID protein aas^((b) of ss^((a)CBM (aa-aa) included^((b) included^((b) NO TBH1 464 26 Yes 27-331 508864.62 6 TBH2 464 26 Yes 27-331 50904 4.67 8 TBH3 464 26 Yes 27-331 508484.67 10 TBH4 464 26 Yes 27-331 50957 4.67 12 TBH5 464 26 Yes 27-33150901 4.67 14 TBH6 464 26 Yes 27-331 50919 4.72 16 TBH7 464 26 Yes27-331 50814 4.67 18 TBH8 464 26 Yes 27-331 50833 4.62 20 TBH9 464 26Yes 27-331 50800 4.62 22 TBH10 464 26 Yes 27-331 50949 4.72 24 TBH11 46426 Yes 27-331 50935 4.72 26 ^((a)The prediction on the signal sequencewas made using the program SignalP v3.0, NN/HMM (Nielsen et al., 1997;Nielsen & Krogh, 1998; Bendtsen et al., 2004). ^((b)The predicted signalsequence was not included. The prediction was made using Clone ManagerProfessional version 9 for Windows, Sci-Ed Software.

Example 3 Production of Recombinant Mannanase Variant Proteins inTrichoderma reesei

Expression plasmids were constructed for production of recombinantmannanase TBH1-TBH11 (SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24and 26) proteins in Trichoderma reesei. The expression plasmidsconstructed are listed in Table 4. The recombinant mannanase geneswithout their own signal sequences were fused to the T. reeseicel7A/cbh1 promoter with T. reesei cel6A/cbh2 CBM carrier and linkerfollowed by Kex2 protease recognition site. The transcriptiontermination was ensured by the T. reesei cel7A/cbh1 terminator and theA. nidulans amdS marker gene was used for selection of the transformantsas described in Paloheimo et al. (2003). The linear expression cassettes(FIG. 1) were isolated from the vector backbones after NotI digestionsand were transformed into T. reesei protoplasts. The host strains used,do not produce any of the four major T. reesei cellulases (CBHI, CBHII,EGI, EGII). The transformations were performed as in Penttilä et al.(1987) with the modifications described in Karhunen et al. (1993),selecting acetamidase as a sole nitrogen source (amdS marker gene). Thetransformants were purified on selection plates through single conidiaprior to sporulating them on PD.

TABLE 4 The expression cassettes constructed to produce TBH1-TBH11 (SEQID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26), recombinantproteins in Trichoderma reesei. The overall structure of the expressioncassettes was as described in FIG. 1. Mannanase protein Expressionplasmid Expression cassette^((a) TBH1 pALK4415 7.5 kb NotI TBH2 pALK44167.5 kb NotI TBH3 pALK4417 7.5 kb NotI TBH4 pALK4418 7.5 kb NotI TBH5pALK4419 7.5 kb NotI TBH6 pALK4420 7.5 kb NotI TBH7 pALK4421 7.5 kb NotITBH8 pALK4422 7.5 kb NotI TBH9 pALK4423 7.5 kb NotI  TBH10 pALK4430 7.5kb NotI  TBH11 pALK4431 7.5 kb NotI ^((a)The expression cassette for T.reesei transformation was isolated from vector backbone by using NotIdigestions.

The mannanase production of the transformants was analyzed from theculture supernatants of the shake flask cultivations. The transformantswere inoculated from the PD slants to shake flasks containing 50 ml ofcomplex lactose-based cellulase inducing medium (Joutsjoki at al. 1993)buffered with 5% KH₂PO₄. The mannanase protein production of thetransformants was analyzed from culture supernatants after growing themfor 7 days at 30° C., 250 rpm Heterologous production of recombinantproteins was analyzed by SDS-PAGE with subsequent Coomassie staining.The supernatants were recovered also for application tests bycentrifugation.

Example 4 Assay of Galactomannanase Activity by DNS-Method

Mannanase activity (MNU) was measured as the release of reducing sugarsfrom galactomannan (0.3 w/w-%) at 50° C. and pH 7.0 in 5 min. The amountof released reducing carbohydrates was determined spectrophotometricallyusing dinitrosalicylic acid.

Substrate (0,3 w/w-%) used in the assay was prepared as follows: 0.6 gof locust bean gum (Sigma G-0753) was in 50 mM sodium citrate buffer pH7 (or citrate phosphate buffer pH 7) at about 80° C. using a heatingmagnetic stirrer and heated up to boiling point. The solution was cooledand let to dissolve overnight in a cold room (2-8° C.) with continuousstirring and insoluble residues were removed by centrifugation. Afterthat solution was filled up to 200 ml by buffer. Substrate was stored asfrozen and melted by heating in a boiling water bath to about 80° C.,cooled to room temperature and mixed carefully before use.

DNS reagent used in the assay was prepared by dissolving 50 g of3.5-dinitrosalisylic acid (Sigma D-550) in about 4 liter of water. Withcontinuous magnetic stirring 80.0 g of NaOH was gradually added and letto dissolve. An amount of 1500 g of Rochelle Salt (K-Na-tartrate, Merck8087) was added in small portions with continuous stirring. The solutionthat was cautiously warmed to a maximum temperature of 45° C., wascooled to room temperature and filled up to 5000 ml. After that it wasfiltered through Whatman 1 filter paper and stored in a dark bottle atroom temperature.

The reaction was first started by adding 1.8 ml of substrate solution toeach of the two test tubes and let to equilibrate at 50° C. for 5minutes, after which 200 μl of suitably diluted enzyme solution wasadded to one of the tubes, mixed well with vortex mixer and incubatedexactly for 5 min at 50° C. Enzyme blanks didn't need to be equilibratedor incubated. The reaction was stopped by adding 3.0 ml of DNS reagentinto both tubes and mixed. 200 μl of sample solution was added to theenzyme blank tubes. Both tubes were placed in a boiling water bath.After boiling for exactly 5 minutes, the tubes were placed in a coolingwater bath and allow them to cool to room temperature. The absorbance ofsample was measured against the enzyme blank at 540 nm and activity wasread from the calibration curve and multiplied by the dilution factor. Asuitable diluted sample yielded an absorbance difference of 0.15-0.4.

Standard curve was prepared 20 mM from mannose stock solution bydissolving 360 mg of mannose (SigmaM-6020, stored in a desiccator) inassay buffer and diluted to solutions containing 3, 6, 10 and 14 μmol/mlof mannose. Standards were handled like the samples except forincubating at 50° C. The absorbances were measured against the reagentblank (containing buffer instead of standard dilution of mannose) at 540nm. Calibration curve was constructed for every series of assays.

One mannanase unit (MNU) was defined as the amount of enzyme thatproduces reductive carbohydrates having a reductive power correspondingto one nmol of mannose from galactomannan in one second under the assayconditions (1 MNU=1nkat).

Example 5 Specific Activities of the Purified Mannanase Variants

Cells and solids were removed from the fermentation culture medium bycentrifugation for 10 min, 4000 g at 4° C. The supernatant of 10 ml wasused for protein purification. The sample was filtered through 0.44 μmPVDF membrane (Millex-HV, Merck Millipore Ltd, Carrigtwohill, IRL). Thefiltrate was loaded onto a HiPrep 26/10 Desalting column (GE Healthcare,Uppsala, Sweden) equilibrated in 20 mM HEPES pH 7. The desalted samplewas then loaded onto a 5 ml HiTrap Q HP column (GE Healthcare, Uppsala,Sweden) pre-equilibrated with 20 mM HEPES pH 7. After sample loading,the column was washed with the same buffer for 20 ml. Proteins wereeluted with linear salt gradient 20 mM HEPES, 500 mM NaCl pH 7 in 15CVs. Fractions of 5 ml were collected and analyzed on SDS-PAGE. Thefractions containing target protein were combined and concentrated to 2ml using Vivaspin 20, 10 kDa MWCO ultrafiltration devices (GEHealthcare). The concentrated sample was further fractionated usingSuperdex 75 26/60 gel-filtration column equilibrated with 20 mM MES, 200mM NaCl pH 6.5. Fractions of 2 ml were collected and analyzed bySDS-PAGE. Fractions containing pure mannanase were combined.

Purified samples were above 95% pure.

Enzyme content of the purified sample was determined using UV absorbance280 nm measurements. Excitation coefficients for each mannanases werecalculated on the bases of amino acid sequence of the enzyme by usingExPASy (Server http://web.expasy.org/protparam/). (Gasteiger et al.2005).

The enzyme activity (MNU) of purified samples was measured as release ofreducing sugars as described in Example 4.

The specific activity (MNU/mg) of mannanases was calculated by dividingMNU activity of purified sample with the amount of purified enzyme.Relative specific activity (%) was calculated by dividing specificactivity of variant mannanase by specific activity of wild type Man7mannanase.

According to 3D model of wild type Man7 potential glycosylation siteasparagine 283 locates near the catalytic center of enzyme.Glycosylation of asparagine 283 could lead to the reduced catalyticactivity of enzyme. To improve the specific activity of mannanasethreonine 285 was mutated to alanine. This mutation preventsN-glycosylation of asparagine 283.

The results indicated that the specific activity of the wild type Man7enzyme produced in Trichoderma was increased more than 10 fold when T285was mutated to alanine (TBH5, TBH6, TBH9 and TBH11) (FIG. 2). On theother hand, specific activity of the TBH10 variant, in whichglycosylation sites other than N283 were eliminated, was not improved(FIG. 2).

The results clearly indicated that the mutation which preventglycosylation of N283 improves the specific activity of mannanasevariant while eliminating other glycosylation sites do not showsignificant effects on specific activity. Production yield (measured asmannanase activity) was higher with TBH5, TBH6, TBH9 and TBH11 (data notshown) compared to wild type Man7.

Mannanase variants with higher specific activity result to the decreasedproduction costs and therefore are commercially more feasible.

Example 6 Stain Removal Performance of Mannanase Variants Produced inTrichoderma with Commercial Detergents

Cultivation supernatants of TBH1-TBH11 variants produced in Trichoderma(as described in Example 3) were tested for their ability to removemannanase sensitive standard stains at 40° C. and water hardness of16°dH with commercial detergents and compared to wild type Man7 enzyme.The following artificially soiled test cloths from Center fortestmaterial B.V. (the Netherlands) were used: Chocolate puddingmannanase sensitive on cotton (E-165), Locust bean gum, with pigment oncotton (C-S-73) and Guar gum with carbon black on cotton (C-S-43). Thefabric was cut in 6 cm×6 cm swatches and 2 pieces of each were used intest.

Commercial heavy duty liquid detergent A containing all other enzymesexcept mannanase was used at concentration of 4.4 g per liter of washliquor, Commercial Color detergent powder without enzymes was used at3.8 g/I and Commercial bleach detergent powder without enzymes was usedat 4.2 g/I. Detergent containing wash liquors we prepared in synthetictap water with hardness of 16 °dH. Protease Savinase® 16 L (0.5 w/w %)and amylase Stainzyme® 12 L (0.4 w/w %) was added into hard water usedwith commercial color and bleach detergent powders, the liquid detergentalready contained amylase and protease. pH of the wash liquor of liquiddetergent was approximately 8.3, with color detergent powder approx. 10and with the bleach detergent approx. 9.5.

Mannanases were dosed as 0.025 and/or 0.05 MNU activity per ml of washliquor. Activity was measured as described in Example 4. Control samplecontained the detergent solution but no mannanase.

For synthetic tap water with hardness of 16°dH the following stocksolutions were prepared in deionized water (Milli-Q or equivalent):

Stock solution with 1000°d Calcium-hardness: CaCl2×2 H2O (1.02382.1000,Merck KGaA, Germany) 26,22 g/l

Stock solution with 200°d Magnesium-hardness: MgSO4×7 H2O (1.05886.1000,Merck KGaA, Germany) 8,79 g/l H2O

NaHCO3 stock solution: NaHCO3 (1.06329.1000 Merck KGaA, Germany) 29,6g/l 13,3 ml CaCl2 solution, 13,3 ml MgSO4 solution and 10,0 ml offreshly made NaHCO3 solution were added in volumetric flask in the givenorder, made up to 1 liter with deionized water and mixed. The hardnessof water was determined by complexometric titration and found correct.

Stain removal treatments were performed in Atlas LP-2 Launder-Ometer asfollows. Launder-Ometer was first preheated to 40° C. Then detergent,250 ml synthetic tap water with hardness of 16°dH and diluted enzyme(<1,0 ml) were added into 1,2 liter containers. Stains were added andthe Launder-Ometer was run at 40° C. for 60 min with a rotation speed of42 rpm. After that the swatches were carefully rinsed under runningwater and dried overnight at indoor air, on a grid protected againstdaylight.

The stain removal effect was evaluated by measuring the colour asreflectance values with Konica Minolta CM-3610A spectrophotometer usingL*a*b* color space coordinates (illuminant D65/10°, 420 nm cut). Fadingof the stains, indicating mannanase performance (stain removalefficiency) was calculated as ΔL* (delta L*), which means lightnessvalue L* of enzyme treated fabric minus lightness value L* of fabrictreated with washing liquor without mannanase (control). Final results(total stain removal effect) were shown as sum of ΔL* of each stains.Color values of each stains were average of 2 swatches.

The results obtained with commercial liquid detergent are shown in FIGS.3A and B, the results with commercial color detergent powder in FIGS. 4Aand B and the results with commercial bleach detergent in FIG. 5. Allvariants (TBH1-TBH11) showed excellent stain removal performance withcommercial detergents of different types. Performance of variants wassimilar to wild type.

Example 7 Efficiency Study with Mannanase Alone and in Combination witha Non-Starch Polysaccharide (NSP) Degrading Enzyme in Broilers

Effects of recombinant mannanase variants of the invention are studiedon growth in broilers. Ultrafiltrate of the fermentation broth includinga recombinant mannanase variant is dried and target levels applied to apelleted broiler diet alone or in combination with a commercialavailable xylanase based product.

A control diet based on corn and dehulled solvent extracted soybean mealis fed without enzyme or added by different levels of the recombinantmannanase variants of the invention alone or in combination with astandard dose of a commercial xylanase.

Initial weight of the broilers is between 30 g and 50 g. The trial lastsbetween three and five weeks. Each treatment consists at minimum of sixreplicates with 10 broilers each. In each case the diet is analysed formoisture, crude protein, crude fibre, fat, ash, and enzyme protein.

Five diets are prepared:

-   -   1) unsupplemented control (BD)    -   2) BD+mannanase 1−500 mg/kg    -   3) BD+mannanase 1−1000 mg/kg    -   4) BD+mannanase 1−500 mg/kg+xylanase 1−10 mg/kg    -   5) BD+xylanase 1−10 mg/kg

Health status and mortality of the animals is checked daily by visualinspection. At days 0, 14, and 35 body weight gain (BW), feed intake(FI), and feed-conversion ratio (FCR) are measured. FCR is calculated asthe total feed consumed divided by the weight gain during the sameperiod. Determination of the effect of the recombinant mannanasevariants is based on the comparison to those animals fed the same dietor the same diet but added by xylanase.

Example 8 Instant Coffee Production

Pure mannan is the main storage polysaccharide component of coffeeendosperms and is responsible for their high viscosity, which negativelyaffects the technological processing of instant coffee and increasesenergy consumption during drying. Those effects are attributed to mannanforming hard, insoluble crystalline structures. β-mannanase, oftentogether with other enzymes such as pectinase and cellulase, is addedduring the concentration step of instant coffee production to reduceviscosity in coffee extracts. Mannanase is also be employed forhydrolyzing galactomannans present in a liquid coffee extract in orderto inhibit gel formation during freeze drying of instant coffee.Furthermore, due to the use of enzymatic treatment the coffee beanextracts can be concentrated by a low cost procedure such asevaporation.

The test is performed according the following flow-chart of FIG. 6 attemperatures of 10° C. and an enzyme dosage of 0.15% d.s.

Mannanase variants of the invention are tested in mixture composed ofdifferent enzymes, such as pectinases and cellulases.

The viscosity of the coffee extract increases significantly over timeunder standard process conditions. However, the viscosity issignificantly reduced using the enzyme mixture containing the mannanasevariants of the invention resulting an improved downstream processingsuch as spray- or freeze drying.

Example 9 Pineapple Processing

In particular, mannanase is useful for pineapple mill juice extractionand clarification, as pineapple contains a large fraction of mannans,including glucomannans and galactomannans.

Mannanase helps to improve extraction of valuable fruit components,lower the viscosity of fruit juice prior to concentration, and increasefiltration rate and stability of the final product.

The pineapples are crushed in a meat grinder and fill 500 g mash in a1000 ml beaker. The enzyme is applied at 21° C. with a reaction time of60 minutes. The mash is then pressed with a small Hafico press accordingto the press protocol: 0 bar 2 min-50 bar 2 min-100 bar 2 min-150 bar 2min-200 bar 1 min-300 bar 1 min-400 bar 1min. The obtained juice is thencentrifuged at 4500 rpm for 5 minutes and analyzed for turbidity andviscosity.

Mannanase variants of the invention are tested in enzyme mixtures A, Band C (Table 5).

The enzymes are first diluted with tab water before being added to thepineapple mash.

TABLE 5 Enzyme mixtures Enzyme Dosage 5 ml of activity [ppm] [% enzymesolution] blank 5 ml H2O Mixture A Pectinase 50 0.50% Mixture BPectinase + 50 0.50% Arabanase Mixture C Pectinase + 50 0.50% Mannanase

Applying mannanase variants of the invention leads to increased yieldand lower turbidity of juice in pineapple processing.

Example 10 Mannanase Treatment of Soya Beans for Soya Milk Production

For the enzymatic treatment of soya beans to get soya milk the “hotprocess” is commonly used. For the hot soya milk process the dried soyabeans were mixed and crushed in a mixer with boiling tap water in aratio of 1:7 (soaked beans: water). The whole soya slurry is cooled downto 50-55° C. before enzyme addition. The pH level for the soya slurryshould be around pH 6.5 and can be adjusted with NaHCO3. The mannanaseenzyme is dosed at 1 kg/t of dried soya beans into the slurry andstirred for 30 min. After completion of the reaction time, the slurry ispressed using a laboratory press to obtain the final product: soya milk.In order to ensure the same pressing profile, the pressure as well asthe corresponding pressing time is specified, as shown in Table 6.Besides the sample for enzymatic reaction, a control sample without anyenzyme is prepared, in which the enzyme solution was replaced withwater.

TABLE 6 Press scheme pressure [bar] 0 50 100 300 time [min] 2 2 2 1

After pressing the soya milk is heated in a microwave until boiling tostop the enzyme reaction. Analysis of the soya milk:

-   -   Yield in gram/time    -   °Brix, which gives a direct correlation of the amount of sugar        in the soy milk, is determined with a refractometer    -   The turbidity of the juice is measured with a NTU-photometer,        which measures the nephelometric turbidity.    -   The brightness will be measured with a LAB-measurement    -   Protein content is determined with a CN-Analyser (combustion        method)    -   Flavour

Soya milk treated with the mannanase variants of the invention shows aincreased yield, brighter colour, increased °Brix, a lower turbidity, ahigher protein content and a better taste (off flavour removal).

Without limiting the scope and interpretation of the patent claims,certain technical effects of one or more of the aspects or embodimentsdisclosed herein are listed in the following: A technical effect isdegradation or modification of mannan. Another technical effect isprovision of mannanase which has good storage stability.

The foregoing description has provided by way of non-limiting examplesof particular implementations and embodiments of the invention a fulland informative description of the best mode presently contemplated bythe inventors for carrying out the invention. It is however clear to aperson skilled in the art that the invention is not restricted todetails of the embodiments presented above, but that it can beimplemented in other embodiments using equivalent means withoutdeviating from the characteristics of the invention.

Furthermore, some of the features of the above-disclosed aspects andembodiments of this invention may be used to advantage without thecorresponding use of other features. As such, the foregoing descriptionshould be considered as merely illustrative of the principles of thepresent invention, and not in limitation thereof. Hence, the scope ofthe invention is only restricted by the appended patent claims.

In an embodiment at least one component of the compositions of theinvention has a different chemical, structural or physicalcharacteristic compared to the corresponding natural component fromwhich the at least one component is derived from. In an embodiment saidcharacteristic is at least one of uniform size, homogeneous dispersion,different isoform, different codon degeneracy, differentpost-translational modification, different methylation, differenttertiary or quaternary structure, different enzyme activity, differentaffinity, different binding activity, and different immunogenicity.

REFERENCES

Bendtsen J D, Nielsen H, von Heijne G, and Brunak S. (2004) Improvedprediction of signal peptides: SignaIP 3.0. J. Mol. Biol. 340:783-795.

Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M. R., AppelR. D., Bairoch A.;Protein Identification and Analysis Tools on theExPASy Server; (In) John M. Walker (ed): The Proteomics ProtocolsHandbook, Humana Press (2005). pp. 571-607.

Joutsjoki V V, Torkkeli T K, and Nevalainen K M H. (1993) Transformationof Trichoderma reesei with the Hormoconis resinae glucoamylase P (gamP)gene: production of a heterologous glucoamylase by Trichoderma reesei.Curr. Genet. 24: 223-228.

Karhunen T, Mäntylä M, Nevalainen K M H, and Suominen P L. (1993) Highfrequency one-step gene replacement in Trichoderma reesei. I.Endoglucanase I overproduction. Mol. Gen. Genet. 241: 515-522.

Nielsen H, Engelbrecht J, Brunak S, and von Heijne G. (1997)Identification of prokaryotic and eykaryotic signal peptides andprediction of their cleavage sites. Protein. Eng. 10: 1-6.

Nielsen H, and Krogh A. (1998) Prediction of signal peptides and signalanchors by a hidden Markov model. In: Proceedings of the SixthInternational Conference on Intelligent Systems for Molecular Biology(ISMB 6), AAAI Press, Menlo Park, Calif., p. 122-130.

Paloheimo M, Mäntylä A, Kallio J, and Suominen P. (2003) Highyieldproduction of a bacterial xylanase in the filamentous fungus Trichodermareesei requires a carrier polypeptide with an intact domain structure.Appl. Env. Microbiol. 69: 7073-7082.

Penttilä M, Nevalainen H, Rättö M, Salminen E, and Knowles J. (1987) Aversatile transformation system for the cellulolytic filamentous fungusTrichoderma reesei. Gene 61: 155-164.

1. A variant of mannanase having mannanase activity and anon-glycosylated amino acid at the position 283, wherein the variant isselected from the group consisting of 1) a polypeptide having at least85% sequence identity to residues 27-331 of SEQ ID NO: 2; 2) apolypeptide encoded by a polynucleotide that hybridizes under highstringency conditions with a) nucleotides 79-993 of SEQ ID NO: 1 (man 7)b) the full-length complement of a); and 3) a variant encoded by apolynucleotide having at least 95% sequence identity to the SEQ ID NO: 1or the genomic DNA sequence thereof; and wherein the amino acidnumbering corresponds to the amino acid numbering of SEQ ID NO: 2 (Man7)full length amino acid sequence containing a signal sequence.
 2. Thevariant of claim 1, wherein at least position N283, T285, or S285,preferably T285 or S285, most preferably T285, is substituted by aresidue which prevents N-linked glycosylation of the residue 283 whenexpressed in a host cell capable of N-linked glycosylation.
 3. Thevariant of claim 1, wherein the position T285 or S285 is substituted toa residue other than T or S.
 4. An enzyme composition comprising thevariant of mannanase of claim 1 and a. at least one preservativeselected for example from group consisting of organic acid, citric acid,ascorbic acid, benzoic acid and their salts and derivatives, sodiumbenzoate, benzoate, hydroxybenzoate and derivatives, sorbic acid, sodiumsorbate, sorbate, salts, such as sodium chloride or potassium chloride,1,2-Benzisothiazolin-3-one (BIT), or a combination thereof; b.optionally at least one stabilizer selected from polyol, propyleneglycol, polyethylene glycol, hexylene glycol, glycerol, a sugar, sugaralcohol, polysaccharide, lactic acid, boric acid, boric acid derivative,aromatic borate ester, 4-formylphenyl boronic acid, phenyl boronic acidderivative, peptide, surfactant, or a combination thereof; c. optionallyat least one enzyme selected from proteases, amylases, cellulases,lipases, xylanases, mannanases, cutinases, esterases, phytases, DNAses,pectinases, pectinolytic enzymes, pectate lyases, carbohydrases,arabinases, galactanases, xanthanases, xyloglucanases, laccases,peroxidases and oxidases with or without a mediator, or a combinationthereof; and d. optionally at least one filler selected frommaltodextrin, flour, sodium chloride, sulfate, sodium sulfate, or acombination thereof.
 5. The enzyme composition of claim 4 in the form ofa liquid composition or a solid composition such as solution,dispersion, paste, powder, granule, granulate, coated granulate, tablet,cake, crystal, crystal slurry, gel, or pellet.
 6. A detergentcomposition comprising the variant of mannanase of any one of claim 1 orthe enzyme composition of claims 4-5.
 7. The detergent composition ofclaim 6 in a form of a liquid detergent or a solid detergent preferablyin a form of a bar, a homogenous tablet, a tablet having two or morelayers, a pouch having one or more compartments, a regular or compactpowder, a granule, a granulate, a paste, a gel, or a regular, compact orconcentrated liquid.
 8. The detergent composition of claim 6 furthercomprising one or more additional enzyme selected from the groupconsisting of protease, lipase, cutinase, amylase, carbohydrase,cellulase, pectinase, pectatelyase, pectinolytic enzyme, esterase,mannanase, arabinase, galactanase, xylanase, oxidase, xanthanase,xyloglucanase, laccase, DNAse and/or peroxidase, preferably selectedfrom the group consisting of proteases, amylases, cellulases andlipases.
 9. A recombinant host cell comprising genetic elements thatallow producing at least recombinant polypeptide comprising the variantof mannanase of claim
 1. 10. The recombinant host cell of 9, wherein thehost cell is selected from the group consisting of: fungal cells,filamentous fungal cells from Division Ascomycota, SubdivisionPezizomycotina; preferably from the group consisting of members of theClass Sordariomycetes, Subclass Hypocreomycetidae, Orders Hypocrealesand Microascales and Aspergillus, Chrysosporium, Myceliophthora andHumicola; more preferably from the group consisting of FamiliesHypocreacea, Nectriaceae, Clavicipitaceae, Microascaceae, and GeneraTrichoderma (anamorph of Hypocrea), Fusarium, Gibberella, Nectria,Stachybotrys, Claviceps, Metarhizium, Villosiclava, Ophiocordyceps,Cephalosporium, and Scedosporium; more preferably from the groupconsisting of Trichoderma reesei (Hypocrea jecorina), T. citrinoviridae,T. longibrachiatum, T. virens, T. harzianum, T. asperellum, T.atroviridae, T. parareesei, Fusarium oxysporum, F. gramineanum, F.pseudograminearum, F. venenatum, Gibberella fujikuroi, G. moniiiformis,G. zeaea, Nectria (Haematonectria) haematococca, Stachybotrys chartarum,S. chlorohalonata, Claviceps purpurea, Metarhizium acridum, M.anisopliae, Villosiclava virens, Ophiocordyceps sinensis, Acremonium(Cephalosporium) chrysogenum, and Scedosporium apiospermum, andAspergillus niger, Aspergillus awamori, Aspergillus oryzae,Chrysosporium lucknowense, Myceliophthora thermophila, Humicolainsolens, and Humicola grisea, bacterial cells, preferably gram positiveBacilli such as B. subtilis, B. licheniformis, B. megaterium, B.amyloliquefaciens, B. pumilus, gram negative bacteria such asEscherichia coli, actinomycetales such as Streptomyces sp., and yeasts,such as Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica,most preferably Trichoderma reesei.
 11. The recombinant host cell ofclaim 9, wherein the recombinant polypeptide is a fusion protein, whichfurther comprises at least one of: an amino acid sequence providing asecretory signal sequence; an amino acid sequence which facilitatespurification, such as an affinity tag or His-tag; an amino acid sequencewhich enhances production, such as an amino acid sequence which is acarrier, such as CBM; an amino acid sequence having an enzyme activity;and an amino acid sequence providing for the fusion protein with bindingaffinity, such as a carbohydrate binding moiety.
 12. A method forproducing a recombinant polypeptide having mannanase activitycomprising: a. cultivating a recombinant host cell of claim 9, whereinthe genetic elements comprise at least one control sequence whichcontrols the production of the recombinant polypeptide in therecombinant host cell; the genetic elements optionally comprise at leastone sequence encoding a signal sequence for transporting the recombinantpolypeptide outside the host cell; and cultivating is carried out inconditions allowing production of the recombinant polypeptide; and a.recovering the recombinant polypeptide.
 13. A method for degrading ormodifying mannan containing material comprising treating said mannancontaining material with an effective amount of the enzyme compositionof claim
 4. 14. The method of claim 13 wherein the mannan containingmaterial is plant based material, textile, waste water, sewage, oil, ora combination thereof.
 15. An animal feed comprising the enzymecomposition of claim 4, and at least one protein source of plant originor a mannan containing product or by-product, and a. Optionally at leastone enzyme selected from protease, amylase, phytase, xylanase,endoglucanase, beta-glucanase, or a combination thereof; and b.Optionally at least one filler selected from maltodextrin, flour, salt,sodium chloride, sulfate, sodium sulfate, or a combination thereof. 16.A feed supplement comprising the enzyme composition of claim 4; and a.Optionally at least one enzyme selected from protease, amylase, phytase,xylanase, endoglucanase, beta-glucanase, or a combination thereof; andb. Optionally at least one filler selected from maltodextrin, flour,salt, sodium chloride, sulfate, sodium sulfate or a combination thereof.17. A use of the variant of claim 1 in oil drilling or hydro-fracturing.18. A use of the variant of claim 1 in processing coffee extract, fruitjuice, pineapple juice, or soya milk.